Bottom Line:
Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts.By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling.Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling.

ABSTRACTCell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.

fig7: Raft targeting of LCK-SHP2 triggers 397Y-FAK phosphorylation in nonadhered cells. (A) Equal amounts of lysates from serum-starved cytSHP-2, SRC-SHP2, LCK-SHP2, or LCK-SHP2C/S cells detached (sus) and replated on Fn for 5 (att) or 30 min (spr) were precipitated with anti-FAK antibody. Pellets were probed with anti–397Y-FAK, –861Y-FAK and –FAK antibodies. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 were transfected in 293T cells or in 293T cells stably expressing FRNK. Equal amounts of lysates from nonattached (sus) and Fn-replated for 5 (att) or 30 min (spr) were probed with anti–397Y-FAK, -FAK, –phospho-ERK and -ERK antibodies. Anti-HA antibody was used to control FRNK expression.

Mentions:
FAK phosphorylation analysis using anti-phosphospecific antibodies showed a significant 397Y-FAK fraction in nonattached LCK-SHP2 cells, but not in cytSHP-2 or SRC-SHP2 cells (Fig. 7 A). FAK phosphorylation kinetics in mock-transfected cells is similar to that in cytSHP-2 or SRC-SHP2, again indicating that the effect is not due to SHP-2 overexpression. LCK-SHP2C/S expression abrogates 397Y-FAK phosphorylation in nonattached cells (Fig. 7 A), indicating that the LCK-SHP2 effect requires PTP activity. Basal FAK phosphorylation in LCK-SHP2 cells is 397Y-specific, as 861Y-FAK is not observed (Fig. 7 A), consistent with the conclusion that SHP-2 partitioning does not affect SFK activity.

fig7: Raft targeting of LCK-SHP2 triggers 397Y-FAK phosphorylation in nonadhered cells. (A) Equal amounts of lysates from serum-starved cytSHP-2, SRC-SHP2, LCK-SHP2, or LCK-SHP2C/S cells detached (sus) and replated on Fn for 5 (att) or 30 min (spr) were precipitated with anti-FAK antibody. Pellets were probed with anti–397Y-FAK, –861Y-FAK and –FAK antibodies. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 were transfected in 293T cells or in 293T cells stably expressing FRNK. Equal amounts of lysates from nonattached (sus) and Fn-replated for 5 (att) or 30 min (spr) were probed with anti–397Y-FAK, -FAK, –phospho-ERK and -ERK antibodies. Anti-HA antibody was used to control FRNK expression.

Mentions:
FAK phosphorylation analysis using anti-phosphospecific antibodies showed a significant 397Y-FAK fraction in nonattached LCK-SHP2 cells, but not in cytSHP-2 or SRC-SHP2 cells (Fig. 7 A). FAK phosphorylation kinetics in mock-transfected cells is similar to that in cytSHP-2 or SRC-SHP2, again indicating that the effect is not due to SHP-2 overexpression. LCK-SHP2C/S expression abrogates 397Y-FAK phosphorylation in nonattached cells (Fig. 7 A), indicating that the LCK-SHP2 effect requires PTP activity. Basal FAK phosphorylation in LCK-SHP2 cells is 397Y-specific, as 861Y-FAK is not observed (Fig. 7 A), consistent with the conclusion that SHP-2 partitioning does not affect SFK activity.

Bottom Line:
Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts.By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling.Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling.

ABSTRACTCell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.